Structural variability of E. coli thioredoxin captured in the crystal structures of single-point mutants

Sci Rep. 2017 Feb 9;7:42343. doi: 10.1038/srep42343.

Abstract

Thioredoxin is a ubiquitous small protein that catalyzes redox reactions of protein thiols. Additionally, thioredoxin from E. coli (EcTRX) is a widely-used model for structure-function studies. In a previous paper, we characterized several single-point mutants of the C-terminal helix (CTH) that alter global stability of EcTRX. However, spectroscopic signatures and enzymatic activity for some of these mutants were found essentially unaffected. A comprehensive structural characterization at the atomic level of these near-invariant mutants can provide detailed information about structural variability of EcTRX. We address this point through the determination of the crystal structures of four point-mutants, whose mutations occurs within or near the CTH, namely L94A, E101G, N106A and L107A. These structures are mostly unaffected compared with the wild-type variant. Notably, the E101G mutant presents a large region with two alternative traces for the backbone of the same chain. It represents a significant shift in backbone positions. Enzymatic activity measurements and conformational dynamics studies monitored by NMR and molecular dynamic simulations show that E101G mutation results in a small effect in the structural features of the protein. We hypothesize that these alternative conformations represent samples of the native-state ensemble of EcTRX, specifically the magnitude and location of conformational heterogeneity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkylation
  • Crystallography, X-Ray
  • Cysteine / genetics
  • Escherichia coli / metabolism*
  • Magnetic Resonance Spectroscopy
  • Molecular Dynamics Simulation
  • Mutant Proteins / chemistry
  • Mutation / genetics*
  • Osmolar Concentration
  • Oxidation-Reduction
  • Protein Conformation
  • Thioredoxins / chemistry*
  • Thioredoxins / genetics*

Substances

  • Mutant Proteins
  • Thioredoxins
  • Cysteine